Shear stress modulates endothelial KLF2 through activation of P2X4.

Vascular endothelial cells that are in direct contact with blood flow are exposed to fluid shear stress and regulate vascular homeostasis. Studies report endothelial cells to release ATP in response to shear stress that in turn modulates cellular functions via P2 receptors with P2X4 mediating shear stress-induced calcium signaling and vasodilation. A recent study shows that a loss-of-function polymorphism in the human P2X4 resulting in a Tyr315>Cys variant is associated with increased pulse pressure and impaired endothelial vasodilation. Although the importance of shear stress-induced Krüppel-like factor 2 (KLF2) expression in atheroprotection is well studied, whether ATP regulates KLF2 remains unanswered and is the objective of this study. Using an in vitro model, we show that in human umbilical vein endothelial cells (HUVECs), apyrase decreased shear stress-induced KLF2, KLF4, and NOS3 expression but not that of NFE2L2. Exposure of HUVECs either to shear stress or ATPγS under static conditions increased KLF2 in a P2X4-dependent manner as was evident with both the receptor antagonist and siRNA knockdown. Furthermore, transient transfection of static cultures of human endothelial cells with the Tyr315>Cys mutant P2X4 construct blocked ATP-induced KLF2 expression. Also, P2X4 mediated the shear stress-induced phosphorylation of extracellular regulated kinase-5, a known regulator of KLF2. This study demonstrates a major physiological finding that the shear-induced effects on endothelial KLF2 axis are in part dependent on ATP release and P2X4, a previously unidentified mechanism.

In-situ identification of copper-based green pigments on paintings and manuscripts by reflection FTIR.

In this work non-invasive reflection infrared spectroscopy was used to discriminate between different Cu-based green pigments (namely, malachite, verdigris, chrysocolla, emerald green and phthalo green). The pigments, chosen because of their historical widespread use in artworks, were investigated as pure powders and in situ on painted models by reflection FTIR spectroscopy. The distortion arising as a result of the specular and diffuse component of reflected radiation was evaluated as function of the optical and surface properties of the investigated mock-ups. Use of the Kramers-Kronig (KK) algorithm to correct for the distortion arising from the surface reflection gave k index maxima shifted by more than 20 cm(-1) relative to those obtained in conventional transmission mode. These findings stress the need to carefully manage use of the KK algorithm on reflection spectra to avoid erroneous assignment. On the other hand, combination and overtone bands (which are enhanced by the diffuse reflection) were proved to enable reliable and sensitive identification of most of the pigments studied. Knowledge acquired by study of pure compounds and painted models was subsequently applied to interpret the spectra acquired from paintings and manuscripts.

Analysis of weaning-induced stress in Saanen goat kids.

In young ruminants' life, weaning often coincides with a period of growth stasis and poor welfare. The present study aimed at evaluating the effect of coping with the new diet on behavioural and haematological stress indicators in goat kids subjected to a commonly adopted weaning practice. Immediately after birth, male Saanen goat kids were divided into two groups: MILK and WMIX. All were fed colostrum for the first 3 days and then goat milk to the age of 29 days. After that, MILK kids continued to receive milk, while the WMIX kids underwent weaning and were completely weaned by day 48. Animal behaviour was recorded daily. From day 23-50, blood samples were taken weekly and analysed for indicators of stress and immune function. No abnormal behaviour, such as injurious behaviours or stereotypies, was observed in either of the experimental groups throughout the experimental period. During the last week, fasting plasma cortisol level was significantly lower, whereas plasma activity of both alanine aminotransferase (ALT) and aspartate aminotransferase (AST) was significantly higher in WMIX kids, in relation to the MILK ones. Anyway, data were within the normal physiological range and no difference was observed neither in plasma haptoglobin, ceruloplasmin, albumin and antithrombin III, nor in plasma immunoglobulin A and G, at any time, signalling no stressful condition. Therefore, differences observed in cortisol, ALT and AST could be the consequence of the metabolic changes that occur during the transition from pre-ruminant to ruminant state. The gradual weaning at 48 days of age did not result in any stressful condition and had no negative effect on weight gain. Results suggest that parameters commonly adopted to provide information on animal stress, such as cortisol and aminotransferase activity, can vary in relation to the physiological status of the animals and may bias stress assessment.

Ghrelin, insulin and pancreatic activity in the peri-weaning period of goat kids.

Two groups of 3-day-old Saanen goat kids (MILK and WMIX) were studied in order to investigate the effect of weaning on plasma ghrelin and insulin and on pancreatic activity. MILK kids received goat milk to age 50 days; WMIX kids were initially fed milk but started weaning at 30 days and were completely weaned by 48 days. Dry matter intake and body weights were recorded, and plasma samples were analysed for metabolites, ghrelin, insulin, leptin and α-amylase. At 50 days, all the animals were slaughtered, pancreas samples were analysed for α-amylase, zymogen, DNA and ribosomal capacity (RNA/zymogen). Seven days after the beginning of the weaning program, dry matter intake in the WMIX group began to decrease in relation to the MILK group. Nonetheless, body weight did not differ throughout the study period. Weaning significantly decreased plasma levels of glucose, amino acids, urea and insulin, but increased creatinine and ghrelin. In weaning kids ghrelin secretion may help minimize the negative consequences of the new diet on dry matter intake. Pancreatic zymogen and ribosomal capacity did not differ between the groups, whereas pancreatic amylase activity was over three times higher in MILK than WMIX kids even though the former had no dietary starch. This finding could be a consequence of lower pancreatic secretion of amylase in the MILK group due to the lack of dietary starch, resulting in pancreatic accumulation, but could also be due to higher plasma insulin in the MILK group, suggesting a role for insulin in the functional development of the pancreas in weaning goats.

Non-invasive identification of organic materials in wall paintings by fiber optic reflectance infrared spectroscopy: a statistical multivariate approach.

The aim of this study is to develop a method for the non-invasive and in situ identification of organic binders in wall paintings by fiber optic mid-FTIR reflectance spectroscopy. The non-invasive point analysis methodology was set-up working on a wide set of wall painting replicas of known composition and using statistical multivariate methods, in particular principal component analysis (PCA), for the interpretation, understanding, and management of data acquired with reflectance mid-FTIR spectroscopy. Results show that PCA can be helpful in managing and preliminary sorting of the large amount of spectra typically collected during non-invasive measurement campaigns and highlight further avenues for research. The developed PCA model was finally applied to the case of a Renaissance wall painting by Perugino assessing it predictability as compared to the interpretation of the single spectrum.

Diet influences the content of bioactive peptides in goat milk.

Leptin, insulin, ghrelin, and IGF-I are circulating peptide hormones concerned with energy homeostasis and the regulation of GH axis. They are present in human milk, and are thought to promote neonatal development. The aim of the present study was to detect these substances in goat milk and determine whether their levels can be modified by changing the macronutrient content of the lactating animals' diet. Sixteen Saanen goats in mid-lactation were divided into two balanced groups, one given a diet containing 17% starch (LS) and the other a diet of 33% starch (HS). Eighty days later, leptin, insulin, ghrelin, and IGF-I were determined by human radioimmunoassay kits in plasma before and after feeding, and in sonicated milk centrifuged to remove fat from morning and evening milking. The HS diet was associated with higher plasma and milk insulin and IGF-I, and plasma ghrelin. Leptin, insulin, and ghrelin in milk were two-three times higher than in plasma; milk IGF-I was only 5-20% of plasma level. Plasma insulin correlated positively with plasma IGF-I; morning milk IGFI and insulin correlated positively with morning plasma levels. These findings demonstrate that human immuno-activities of bioactive peptides are present in goat milk, and also that levels of insulin and IGF-I in milk can be altered by changing the macronutrient content of the diet. Further research is required to determine whether these substances can be transferred from the milk to suckling animals and humans, and whether they have biological activity in such animals.

NT-proBNP changes, oxidative stress, and energy status of hypertrophic myocardium following ischemia/reperfusion injury.

N-terminal pro-B-type natriuretic peptide (NT-proBNP) is a sensitive functional marker in heart disease, including left ventricular hypertrophy (LVH) secondary to valvular aortic stenosis (AS). We evaluated the association between NT-proBNP changes, oxidative stress, energy status and severity of LVH in patients with AS. Ten patients undergoing aortic valve replacement for AS were studied. Plasma NT-proBNP concentrations were performed by electroluminescence immunoassay 15min after the induction of anesthesia (t0), before aortic cross-clamping (t1), before clamp removal (t2), 15min after myocardial reperfusion (t3), and 24h after surgery (t4). Heart biopsies were obtained and high energy phosphates (ATP, ADP, AMP) were analyzed by capillary electrophoresis (CE). In plasma samples from the coronary sinus, nitrate plus nitrite (NOx) concentrations were also analyzed by CE. Echocardiographic measurements were acquired and correlations between biochemical markers and severity of AS were assessed. NT-proBNP peaked significantly at t4 (p<0.001). A linear correlation between NT-proBNP values measured at t0 and t4 was found (R(2)=0.89; p<0.001). A negative correlation between NT-proBNP production and phosphorylation potential (ATP/ADP ratio) was observed (R(2)=0.62; p<0.01). NOx values positively correlated with NT-proBNP levels (p<0.01). NT-proBNP inversely correlated with aortic valvular area (r=81, p<0.01), positively correlated with mean (r=0.82, p<0.01) and maximum left ventricle-to-aortic gradients (r=0.80, p<0.01), and with left ventricular mass (r=0.69, p<0.01). NT-proBNP is a useful marker of LVH and severity of AS. It may complement echocardiographic evaluation of patients with AS in identifying the optimum time for surgery.

Nitric oxide generation is associated with an unbalance of protein tyrosine phosphatases during liver transplantation.

Organ dysfunction secondary to ischemia-reperfusion (I/R) injury still represents a major problem in liver transplantation. Apoptosis has been observed in hepatocytes and sinusoidal endothelial cell, following I/R injury and it has been postulated as a contributing factor in ischemia-reperfusion graft dysfunction, involving a complex series of events, as changes of protein tyrosine-kinase phosphorylation. We evaluated hepatic purine metabolites, protein tyrosine phosphatases (PTPs), nitrate plus nitrite levels (NOx), caspase-3 (C-3) activity and DNA fragmentation in the time course of twelve pig orthotopic liver transplantation. Biopsies were taken before explantation (t0), after cold ischemic storage (t1) and 30 min from reperfusion (t2). During the ischemic period we observed a reduction of high energy phosphates and an increase of purine bases; PTP activity was largely increased. At t2 high energy phosphates showed a tendency to increase with respect to t1, with a partial restoration of phosphorylation potential, measured as ATP/ADT ratio. PTP activity was significantly reduced, with a concomitant increase of NOx production and C-3 activity; in a considerable number of cases we observed a sustained DNA fragmentation. We speculate that NOx production could be related to nitrosative stress, which in turn leads to dynamic alteration in PTP balance and cell signalling, regulating the activity of a number of proteins implicated in apoptotic cell death. These findings could be of interest in new potential strategy to prevent and treat I/R injury.

Fiber-optic fourier transform mid-infrared reflectance spectroscopy: a suitable technique for in situ studies of mural paintings.

A prototypical in situ noninvasive study of ancient mural painting materials has been carried out using an easily manageable fiber-optic Fourier transform mid-infrared (mid-FT-IR) reflectance spectrophotometer. The reported object of the study is the Renaissance fresco by Pietro Vannucci, called il Perugino, located in the church of Santa Maria delle Lacrime (1521, Trevi, Perugia Italy). For the first classification and interpretation of infrared spectra, principal components analysis was used. Spectral artifacts due to lacunas, restoration materials, or alteration products have been identified, as well as two different secco refinements bound in a tempera medium. For the characterization of inorganic pigments, mid-FT-IR spectra have been integrated with other data obtained through in situ X-ray fluorescence (XRF) elemental analysis. This complementary noninvasive approach led to the characterization of Perugino's pigments, even in the presence of complex mixtures. The mid-FT-IR noninvasive technique, in combination with XRF, is thus recommended as a valuable first approach for the examination of mural paintings, permitting the assessment of the execution technique as well as contributing to the evaluation of the conservation state.

[Preliminary results of the effects of electromagnetic fields ELF and TAMMEF on human lymphocytes: evaluation of some biological parameters]. / Studio preliminare sugli effetti di campi elettromagnetici, ELF e TAMMEF, nei linfociti umani: valutazione di alcuni parametri biologici.

AIMS: The Authors have evaluated the effects of low frequency electromagnetic fields on human peripheral blood lymphocytes metabolism. MATERIALS AND METHODS: It has been assayed total proteins and the activities of some purine metabolism enzymes after exposure of the cells to sinusoidal ELF fields (100 Hz frequency) or to the new TAMMEF system fields, characterized by variable frequency, intensity and shape of wave. RESULTS: The protein lymphocytes content, increased after both treatments. Instead, the enzyme activities did not vary, with the exception of Myokinase activity, which, respect to the control, increased after ELF field treatment, and slightly decreased after TAMMEF treatment. This preliminary result was interpreted as a variation of the cellular electric charge stability, stimulated by ELF fields, which induce the Myokinase to increase its rate, to rearrange the correct equilibrium, while the TAMMEF field were useless to maintain the cellular electric charge at normal levels. CONCLUSIONS: We interpreted these preliminary results as follow: the ELF fields influence the cellular electric charge stability, so that the cells must increase MK activity to restore the correct equilibrium, while TAMMEF fields are useful to maintain and regulate cellular electric charge. The results obtained by this preliminary study opens interesting prospective to be further investigated.

Purine metabolism in B-cell lymphocytic leukemia: a microarray approach.

B-cell chronic lymphocytic leukemia (B-CLL) is an adult-onset highly heterogeneous malignancy characterized by a cells resistance to apoptosis rather than to highly proliferative cells. In previous research, we evidenced an imbalance of purine metabolism in B-CLL cells. Since the extracellular adenosine has been proved to induce apoptosis via A2b receptor, enzymes involved in adenosine metabolism could play an important role in apoptosis resistance of B-CLL cells. We prepared a microarray chip for the analysis of 50 selected genes that could be of interest in B-CLL: enzymes of purine de-novo, salvage and catabolic pathway, oxidative stress enzymes, and apoptotis-related proteins. Preliminary results identify many genes of purine metabolism that exhibit low or high expression, while genes involved in signal transduction and apoptosis exhibit lower alterations even if of remarkable interest. This application of microarray technique seems promising and at least a subset of these genes will be valid candidates for further studies.

Non-invasive Investigations of Paintings by Portable Instrumentation: The MOLAB Experience.

The in situ non invasive methods have experienced a significant development in the last decade because they meet specific needs of analytical chemistry in the field of cultural heritage where  artworks are rarely moved from their locations, sampling is rarely permitted, and analytes are a wide range of inorganic, organic and organometallic substances in complex and precious matrices. MOLAB, a unique collection of integrated mobile instruments, has greatly contributed to demonstrate that it is now possible to obtain satisfactory results in the study of a variety of heritage objects without sampling or moving them to a laboratory. The current chapter describes an account of these results with particular attention to ancient, modern, and contemporary paintings. Several non-invasive methods by portable equipment, including XRF, mid- and near-FTIR, UV-Vis and Raman spectroscopy, as well as XRD, are discussed in detail along with their impact on our understanding of painting materials and execution techniques. Examples of successful applications are given, both for point analyses and hyperspectral imaging approaches. Lines for future perspectives are finally drawn.

Purine nucleotide metabolism: specific aspects in chronic lymphocytic leukemia lymphocytes.

The metabolism of purine nucleotides was studied in human peripheral blood lymphocytes from healthy subjects and patients with B-cell chronic lymphocytic leukemia. Nucleotide content was determined by HPLC. The rate of de novo synthesis of purine nucleotides was measured kinetically by following the incorporation of 14C-formate into the nucleotides of a lymphocyte suspension. The patterns of the main enzymes involved in purine nucleotide metabolism (those of the salvage pathway and catabolism) were estimated by a radiochemical method. Although the data expressed in relation to cells and protein showed some discrepancies, several common differences were evident in both cases. The main differences were an increase in NAD and IMP, a sharp decrease in 5'-nucleotidase activities and in total guanylate content and synthesis, and an increase in the A/G ratio in lymphocytes of patients with respect to controls. The changes in these parameters in CLL indicate an imbalance in purine metabolism and may play a specific role in the biology of the leukemia cell. They are also potential biochemical markers of lymphoid malignancies and may be useful in chemotherapic applications.

Restoration of rat liver L-threonine dehydratase activity by pyridoxamine 5'-phosphate: the half-transaminating activity of L-threonine dehydratase and its regulatory role.

When a highly purified preparation of rat liver l-threonine deaminase (l-TDH, EC 4.2.1.16) was 99% inactivated by dialysis, removing bound pyridoxal 5'-phosphate (PLP), the apoenzyme was reactivated not only by PLP but also by pyridoxamine 5'-phosphate (PMP). When purified by HPLC, the commercial PMP used in the incubation mixture was found to contain only extremely small amounts of PLP, which could not account for restoration of l-threonine dehydratase activity. HPLC analysis of the assay mixtures showed that during incubation, sufficient PLP had been formed for reactivation of the apoenzyme. The apoenzyme evidently bound PMP and triggered transamination between PMP and the keto acids, which either contaminated, or were formed by the minimal amount of PLP-holoenzyme always present even in the dialyzed preparation. When sufficient PLP was formed, the PLP-holoenzyme and the original 'true' l-threonine dehydratase activity were restored. When PMP was incubated with the apoenzyme in the presence of small quantities of keto acids (pyruvate or 2-oxobutyrate) small amounts of l-alanine or l-aminobutyrate were formed. The reaction was not reversible; l-alanine and l-aminobutyrate did not react with the PLP-holoenzyme. No transaminating activity occurred with other amino acids. These results show that l-threonine dehydratase exists in two forms: the well known stable apoenzyme-PLP (hydrolase deaminating) and the transient apoenzyme-PMP (non-reversible half-transaminating). Half-transamination has the biological role of keeping the activity of the 'true' l-TDH constant and of regulating intracellular levels of pyruvate, alanine, oxobutyric acid, l-aminobutyric acid, l-threonine and l-serine.

5'-nucleotidase activity in lymphocytes from patients affected by B-cell chronic lymphocytic leukemia.

OBJECTIVES: The activity of membrane-bound ecto-5'-nucleotidase and soluble e-Ns and c-N-II 5'-nucleotidases was evaluated on lymphocytes from patients affected by B-cell chronic lymphocytic leukemia (B-CLL). A statistically significative decrease in ecto-5'-nucleotidase, e-Ns, and c-N-II activities was observed in peripheral blood lymphocytes and in B and T populations from affected individuals. DESIGN AND METHODS: For the assay of ecto-5'-nucleotidase, e-Ns, and c-N-II activity we used a radioactive procedure coupled to HPLC. Since the ecto-5'-nucleotidase is identified as CD73 antigen, we performed immunofluorescence analysis using a specific monoclonal antibody. We analyzed ecto-5'-nucleotidase mRNA by RT-PCR to ascertain the possibility of an alteration in the transcription of its gene. RESULTS: A decrease in ecto-5'-nucleotidase activity was correlated to reduction in ecto-5'-nucleotidase positive cells (CD73+) in leukemia patients. RT-PCR produced a fragment of the expected size and the specific mRNA was found expressed in both healthy subjects and leukemia patients. CONCLUSIONS: The decrease in ecto-5'-nucleotidase activity in patients with B-CLL is not due to loss of transcription of the specific mRNA. The presence of point mutations, splicing alteration, or posttranslational modifications must be investigated. If a defect at DNA or RNA level will be detected, the molecular analysis will be considered for diagnosis of B-cell chronic lymphocytic leukemia.

Methylazoxymethanol-induced microencephaly: persistent increase of cortical somatostatin-like immunoreactivity.

Treatment of pregnant rats with methylazoxymethanol (MAM) at day 15 of gestation induces a marked microencephaly in the offspring, characterized by impaired formation of interneurons in the areas affected. We have measured somatostatin immunoreactivity in the cortex, striatum and hippocampus of the offspring of pregnant rats treated with graded doses of MAM. A long-lasting increase in the cortical levels of this peptide was found, suggesting that somatostatinergic interneurons might be selectively spared by administration of the cytotoxic agent at this gestational age.

Fatty acid composition of phospholipids, triglycerides and cholesterol in serum of castrated and estradiol treated rats.

We have studied the levels of phospholipids, triglycerides, cholesterol esters, and their fatty acid composition in serum for normal, castrated and estradiol treated rats. The sex hormones did not greatly affect the levels of the various lipid fractions which did not undergo great significant variations, under the different treatments. More evident variations occurred in the percent composition of fatty acid and in the content of the various saturated (SAT), unsaturated (UNSAT), essential (EFA) and non essential fatty acids (NEFA). We studied the most important ratios: EFA/NEFA; UNS/SAT; 16:0/16:1; 18:0/18:1, 18:2/18:3; 18:2/20:4. 16:0/16:1; 18:0/18:1 represent the delta9 desaturase, one specific for palmitic, the other for stearic acid. 18:2/18:3 ratio is an index of the delta6 desaturase activity: 18:2/20:4 ratio of delta5 desaturase-elongase. Most changes were evident in triglycerides. We observed a different behaviour of the UNS/SAT and EFA/NEFA ratios in phospholipids and cholesterol esters, which may reflect either an effect of the sex hormones on the exchange of fatty acids between the same lipid fractions, or a redistribution of lipids among different tissues. Great variations were observed of the ratios 16:0/16:1; 18:0/18:1; 18:2/18:3; 18:2/20:4, which are ascribed a different effect of the sex hormones of delta9, delta6, delta5 desaturases.

Behaviour of human lymphocytic isoenzymes of 5'-nucleotidase.

The behaviour of 5'-nucleotidase isoenzymes (ecto-5'-nucleotidase, e-Ns and c-N-II soluble 5'-nucleotidases) was studied in lymphocytes from patients with B-cell chronic lymphocytic leukemia. A strong reduction in ecto- and soluble activities was observed, although the pattern of the three 5'-nucleotidases did not always strictly overlap. A significant decrease (p<0.05) in ecto-5'-nucleotidase, e-Ns and c-N-II was found in B and T populations (B lymphocytes: 1.13, 0.88 and 1.26 nmol/h/10(6) cells versus 95.96, 9.64 and 13.73 nmol/h/10(6) cells in controls; T lymphocytes: 1.31, 0.23 and 0.06 nmol/h/10(6) cells versus 9.25, 1.31 and 2.10 nmol/h/10(6) cells in healthy subjects). The percentage of ecto-5'-nucleotidase-positive cells (CD73+) was reduced in leukemia patients, indicating a lower number of active molecules on the cell surface. The results of RT-PCR analysis showed that the ecto-5'-nucleotidase mRNA of leukemia patients was not defective.

Ecto-5'-nucleotidase in B-cell chronic lymphocytic leukemia.

Literature on the behaviour of ecto-5'-nucleotidase in the course of B-cell chronic lymphocytic leukemia is briefly reviewed and aims for further researches are highlighted.

Determination, activity and biological role of adenylosuccinate lyase in blood cells.

Adenylosuccinate lyase deficiency, which is associated with severe mental retardation and autistic features, was discovered in 1984. Since then this enzyme has been analyzed in many human tissues and it is now generally agreed that screening for this enzyme defect should be performed in all unexplained neurological diseases. The aim of the present study was to analyze adenylosuccinate lyase activity in blood cells by a fast simple method adaptable to screening purposes. The activity was also analyzed in B-lymphocytes from patients with B-cell chronic lymphocytic leukemia. The biological role of adenylosuccinate lyase and its importance in regulating cellular levels of AMP is discussed.